Volume 10, Issue 2, Pages 133-143 (August 2006) The TSC2/mTOR pathway drives endothelial cell transformation induced by the Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor  Akrit Sodhi, Risa Chaisuparat, Jiadi Hu, Amanda K. Ramsdell, Brendan D. Manning, Edward A. Sausville, Earl T. Sawai, Alfredo Molinolo, J. Silvio Gutkind, Silvia Montaner  Cancer Cell  Volume 10, Issue 2, Pages 133-143 (August 2006) DOI: 10.1016/j.ccr.2006.05.026 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 PI3K-dependent phosphorylation of proteins of the Akt/TSC2/mTOR pathway by the KSHV vGPCR A–C: Immunodetection of the levels of P-Akt or Akt (A), phosphorylated Akt substrates (B), and P-TSC2 or TSC2 (C) of COS-7 cells transfected with mammalian vectors encoding for GFP (control), vGPCR, or vGPCR R143A (R143A). Cells were treated with 50 μM LY294002 for 45 min where indicated. D: Immunodetection of the levels of P-Akt, Akt, P-mTOR, mTOR, P-p70 S6K, p70 S6K, P-4EBP1, or 4EBP1 of COS-7 cells transfected with mammalian vectors encoding for GFP (control), vGPCR, or vGPCR R143A (R143A). Cells were treated with 50 μM LY294002 or 50 nM rapamycin (Rap) for 45 min where indicated. Cancer Cell 2006 10, 133-143DOI: (10.1016/j.ccr.2006.05.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Levels of P-Akt and P-S6 ribosomal protein in vGPCR experimental and human KS A: Schematic illustrating the oncogenic potential of vGPCR when specifically expressed in the vascular endothelium of mice using the TIE2-tva transgenic mouse model. B: Representative H&E staining and immunohistochemical detection of the levels of phosphorylated Akt (P-Akt) and phosphorylated S6 ribosomal protein (P-S6) of a Kaposi-like sarcoma formed in the TIE2-tva transgenic mouse model after injection with RCAS-vGPCR (105 IU). The cells demonstrate high cytoplasmic immunoreactivity for both P-Akt and P-S6. Scale bar, 50 μm. C: Representative H&E staining and immunohistochemical detection of the levels of P-Akt and P-S6 of a human AIDS-KS tissue. Most tumor (spindle) cells react with P-Akt and P-S6. Scale bar, 50 μm. Cancer Cell 2006 10, 133-143DOI: (10.1016/j.ccr.2006.05.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Paracrine activation of mTOR by vGPCR A: Phosphorylation of S6 ribosomal protein in murine (SVEC) or human (HMVEC) endothelial cells after 30 min treatment with conditioned media (supernatant) from NIH3T3 stably expressing GFP (control), vGPCR, or the inactive vGPCR mutant (R143A), or conditioned media from 293T cells transfected with mammalian vectors encoding these proteins. Cells were pretreated with 50 nM rapamycin (Rap) for 45 min where indicated. B: Immunodetection of the levels of phospho-S6 ribosomal protein of HMVEC treated for 15 min with IL8 (50 ng/ml), GROα (50 ng/ml), VEGF (5 ng/ml), PDGF (2.5 ng/ml), IL1β (0.0025 ng/ml), IL10 (2.5 ng/ml), IL6 (0.25 ng/ml), TNFα (0.05 ng/ml), IP10 (50 ng/ml), SDF1α (50 ng/ml), or MIG (50 ng/ml). Untreated cells served as a control (control). Cancer Cell 2006 10, 133-143DOI: (10.1016/j.ccr.2006.05.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Activation of the PI3K/Akt pathway by vGPCR induces cell proliferation through the TSC2/mTOR signaling pathway A: Pharmacological inhibition of vGPCR signaling pathways in expressing endothelial cells (EC-vGPCR). Cells were treated with a PI3K inhibitor (LY294002; 1 μM), an mTOR inhibitor (rapamycin [Rap]; 1 nM), an ERK1/2 inhibitor (U0126; 10 μM), a JNK inhibitor (SP600125; 5 μM), or a p38 inhibitor (SB203580; 10 μM). Lysates were analyzed by Western blot using a phospho-antibody for the corresponding kinase substrate. B: Effect of the treatment with increasing doses of LY294002, rapamycin (Rap), U0126, SP600125, or SB203580 on the proliferation of EC-vGPCR. Results are illustrated as percentage of cell proliferation relative to untreated parental (SVEC) control cells. The results are the mean ± SEM of triplicate samples from a single representative experiment that was repeated three times with similar results. C: Schematic demonstrating the regulation of mTOR activity through Akt phosphorylation (and inhibition) of the Rheb GAP, TSC2. Akt-dependent inactivation of TSC2 can be bypassed by overexpressing wild-type Rheb, thereby promoting mTOR activity in the presence of inhibitors of PI3K (e.g., LY294002), but not inhibitors of mTOR (e.g., rapamycin). D: Immunodetection of the levels of AU5-tagged Rheb wild-type, P-mTOR, mTOR, P-p70 S6K, p70 S6K, P-4EBP1, or 4EBP1 in SVECs stably transfected with mammalian vectors encoding for GFP (control) or wild-type Rheb (Rheb). Cells were treated with 50 μM LY294002 or 50 nM rapamycin (Rap) for 45 min where indicated. E: Effects of treatment with increasing doses of LY294002 or rapamycin (Rap) on the proliferation of EC-vGPCR cells or EC-vGPCR cells overexpressing Rheb (EC-vGPCR/Rheb). Results are illustrated as percentage of cell proliferation relative to untreated parental (SVEC) control cells. The results are the mean ± SEM of triplicate samples from a single representative experiment that was repeated three times with similar results. IC50s for each line are shown. Cancer Cell 2006 10, 133-143DOI: (10.1016/j.ccr.2006.05.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Activation of the TSC2/mTOR signaling pathway is required for vGPCR oncogenesis A: Effect of the treatment with an mTOR inhibitor of EC-vGPCR allografts established in athymic nu/nu females. Mice were treated with one cycle (arrows) of either rapamycin (Rap) (10 mg/kg/day) or equivalent volume of vehicle intraperitoneally for 5 consecutive days as described in the Experimental Procedures. The results are expressed as mean tumor weight (mg) ± SEM. Data are from a representative independent experiment that was repeated two times with similar results. B: H&E staining, relative levels of P-S6 ribosomal protein, and BrdU incorporation in representative sections of vehicle- and rapamycin-treated EC-vGPCR tumor tissue. Rapamycin-treated tumors show decreased cellularity, fewer mitotic figures, and decreased BrdU staining, correlating with a marked reduction in P-S6 immunoreactivity. Scale bar, 50 μm. Cancer Cell 2006 10, 133-143DOI: (10.1016/j.ccr.2006.05.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Activation of the TSC2/mTOR signaling pathway is sufficient to transform expressing endothelial cells A: Tumor allografts were established in athymic nu/nu mice by injecting immortalized murine endothelial cells (SVEC) or SVECs expressing vGPCR (EC-vGPCR) or Rheb (EC-Rheb). The results are expressed as mean tumor weight (mg) ± SEM. Data are from a representative independent experiment that was repeated two times with similar results. B: H&E staining and relative levels of P-S6 ribosomal protein in a representative section of EC-Rheb tumor tissue. The tumor is composed of atypical spindle-shaped tumor cells with narrow vascular structures, similar to tumors formed from endothelial cells expressing vGPCR. P-S6 shows cytoplasmic immunoreactivity in most of the tumor cells. Scale bar, 50 μm. Cancer Cell 2006 10, 133-143DOI: (10.1016/j.ccr.2006.05.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Direct activation of mTOR is not sufficient for vGPCR sarcomagenesis in vivo A: Inhibition of proliferation of endothelial cells expressing vGPCR (EC-vGPCR) or coexpressing vGPCR and the rapamycin-resistant mTOR mutant (EC-vGPCR/RR-mTOR) by increasing doses of rapamycin (Rap). Results are illustrated as percentage of cell proliferation relative to untreated parental (SVEC) control cells. The results are the mean ± SEM of triplicate samples from a single representative experiment that was repeated two times with similar results. B: Immunohistochemical staining showing expression of AU1-tagged RR-mTOR in a small number of tumor cells within allografts established in athymic nu/nu females upon injection of EC-vGPCR/RR-mTOR. Scale bar, 50 μm. C: Effect of the treatment with the mTOR inhibitor rapamycin on tumors formed from EC-vGPCR or EC-vGPCR/RR-mTOR. Mice were treated with one cycle (arrows) of either rapamycin (Rap) (10 mg/kg/day) or equivalent volume of vehicle intraperitoneally for 5 consecutive days, as described in the Experimental Procedures. The results are expressed as mean tumor weight (mg) ± SEM. Data are from a representative independent experiment that was repeated once with similar results. Cancer Cell 2006 10, 133-143DOI: (10.1016/j.ccr.2006.05.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 8 vGPCR-expressing mice and TSC2+/− animals develop similar vascular tumors A: Representative H&E staining of a liver hemangioma formed in the TIE2-tva transgenic mouse model after injection with RCAS-vGPCR (105 IU) (vGPCR) or in TSC2-deficient animals (TSC2+/−). Scale bar, 50 μm. B: Gross pathology and representative H&E staining of a Kaposi-like sarcoma formed in the TIE2-tva transgenic mouse model after injection with RCAS-vGPCR (105 IU) (vGPCR) or an angiosarcoma formed in TSC2-deficient animals (TSC2+/−). Scale bar, 50 μm. Cancer Cell 2006 10, 133-143DOI: (10.1016/j.ccr.2006.05.026) Copyright © 2006 Elsevier Inc. Terms and Conditions